This revised research proposal for an NIMH Exploratory/Developmental Grant (R21) will explore the combined use of molecular and cell biological methods with electrophysiological and optical imaging techniques in studies aimed at understanding the cellular mechanisms underlying changes in synaptic efficacy. We propose to apply the use of new molecular tool towards examining the dynamic activity dependent redistribution and insertion of glutamatergic and GABAergic receptors to synapses during development and plasticity. DNA constructs encoding the ionotropic glutamate receptor subunit GluR1 or GABAAalpha 1 receptor subunits fused with the pH-sensitive mutants of enhanced green fluorescent protein ('pHluorins') have been generated, and expressed in cell lines and primary cultures of rat hippocampal neurons. The 'pHluorins' fused to the extracellular NH2-terminal region of the receptors will be examined as sensitive and specific indicators of postsynaptic vesicle fusion-mediated delivery of receptors to the synapse. These experiments designed to optically monitor receptor distributions on the cell surface in living neurons over time are critical for evaluating the hypothesis that redistribution and/ or insertion of postsynaptic ionotropic receptors to activated synapses is a major mechanism underlying changes in synaptic efficacy. Studies proposed are not limited solely to glutamatergic synapses, as we are also interested in whether parallel mechanisms of receptor redistribution play a role in plasticity of GABAergic synapses. The proposed work is a novel and innovative approach to studying the dynamics of synapse formation and synaptic plasticity in living microcircuits of hippocampal neurons in culture. An understanding of fundamental mechanisms underlying synaptic plasticity will provide critical insight to not only physiological processes involved in learning and memory, but also to the etiology of pathophysiological disorders involving the deterioration of cognitive function accompanying mental illness, aging and drug addiction.